Because the majority of mitochondrial proteins are encoded in the nuclear genome, mitochondrial biogenesis and maintenance of mitochondrial homeostasis ultimately depend on nuclear transcription being regulated in response to mitochondrial damage and to changes in cell metabolism or nutrients availability. However, the pathways controlling mitochondria-to-nucleus communication in mammalian cells are still largely unknown. Here, we propose to investigate the hypothesis that the transcriptional cofactor GPS2 regulates mitochondrial homeostasis as a direct mediator of mitochondrial retrograde signaling in mammalian cells with the following Aims: i) Characterize the role of GPS2 in promoting transcriptional activation of nuclear-encoded mitochondrial genes; ii) Dissect the regulation of GPS2 shuttling between mitochondria and nucleus and define the gene programs regulated upon translocation; iii) Elucidate the role of GPS2 retrograde translocation in regulating mitochondrial homeostasis. To achieve these goals, we will use a combination of biochemical techniques, genome wide ChIPseq/RNAseq experiments and open-ended proteomic approaches to dissect the molecular mechanism underlying GPS2-mediated regulation of mitochondrial gene expression in the nucleus and elucidate the regulatory strategies that control GPS2 retrograde translocation both in conditions of mitochondrial stress and during the differentiation of specialized cell lineages. Overall, successful completion of the studies outlined in this application will: 1) identify a novel player in the regulation of mitochondrial gene expression and elucidate the molecular mechanism of its transcriptional activity, 2) dissect the first direct pathway of mitochondrial retrograde signaling in mammalian cells, and 3) reveal an unexpected regulatory strategy for integrating stress and metabolic signaling within the cell through inhibition of non-proteolytic ubiquitination.